It is oftentimes necessary to separate and detect enzymes. This is particularly true, for example, in medical applications where isoenzyme profiles are useful for diagnostic purposes.
Enzymes have heretofore been separated and many techniques have heretofore been suggested and/or utilized to achieve such separation. Examples of separation techniques may be found, for example, in U.S. Pat. Nos. 3,722,181, 3,808,125 and 3,983,299.
Likewise, separated enzymes have been heretofore detected, and detection of such enzymes has normally included incubating the enzyme with an appropriate substrate and then monitoring the formation of the product after a fixed reaction time.
One of the more useful high speed techniques for separating enzymes utilizes liquid chromatographic column technology. More recent advances in this type of separation have made it possible to resolve enzyme mixtures in about ten minutes or less. For example, the five isoenzymes of lactic acid dehydrogenase have been resolved in six minutes and the three isoenzymes of creatine phosphokinase have been resolved in four minutes.
High speed separation of isoenzymes is of particular significance because of the clinical utility of isoenzyme profiles in diagnosing miocardial infarction, pulmonary infarctions, and liver diseases. If coupled into a flow-through system with a dependably fast and accurate enzyme detection system, then high speed liquid chromatography of isoenzymes can become a clinically useful analytical tool.
Heretofore, however, two major problems have been encountered in the design and construction of chromatographic flow-through enzyme detectors. One of these problems is distortion by band spreading of the chromatographic profile in the detector and the other is demixing of enzymes and substrate during passage through the system.
Detectors heretofore known and/or utilized have attempted to overcome these problems but have not been completely successful, at least in many respects. One such attempt, for example, has been made to overcome these problems by utilization of instruments that segment the liquid reactant stream with air bubbles. This alternating gas-liquid segmentation prevents intersegmental mixing while still allowing intersegmental mixing of reactants, but obviously does not permit continuous flow-through of a mixture of separated enzymes and substrate.
Thus, while enzyme detectors have heretofore been suggested, none has been completely successful in providing high performance systems that provide dependably fast and accurate detection.